1. Field of the Invention
This invention utilizes novel heterobifunctional compositions to immobilize polynucleic acids on plastic surfaces. More specifically this invention comprises molecules with hydrophobic regions that can intercalate into plastic and hydrophilic reactive groups that can covalently attach to specific loci on a nucleic acid polymer. Further, these novel nucleic acid surfaces result in new assay technologies.
2. Background Information
The practice of biotechnology, and particularly diagnostics, has increased the demand for products requiring reagent immobilization on substrates. "Reagents" includes proteins, nucleic acids, cells, drugs, and small molecule haptens. Substrates are insoluble matrices for immobilization and can be plastic, glass, silica, carbon, cellulose, or other materials. Plastics are particularly useful substrates as they can be formed into a variety of shapes such as cups, discs, dipsticks, spheres, fibers, tubes, membranes, and particles. Plastics are also used as surface coatings that can be modified further. Additionally, plastics have a high degree of biocompatability, and may be produced of materials having excellent optical properties. Typical plastics useful as substrates include polypropylene, polystyrene, polyethylene, polyvinyl chloride, polysulfone, polycarbonate, cellulose acetate and others. Plastics of styrene, vinyl chloride, acrylic and carbonate are widely used when optical properties are a consideration.
Plastics are often used directly as substrates for immobilization of macromolecules. Polystyrene and polyvinyl chloride will anchor large molecules by electrostatic attraction. However, small molecules require attachment to larger "carrier" molecules before being bound to the plastic. Also, poor direct binding to most plastics limits the use of adsorption immobilization to high surface area systems. For instance, polystyrene latex particles can immobilize far more protein molecules per gram of plastic than molded polystyrene products.
Modification of the plastic surface has been used to increase thee electrostatic interaction and increase the binding of some reagents. Electrostatic interactions alone will immobilize only a limited number of reagents. Detergents introduced in the system can cause reagent loss.
Reagent molecules are typically mobilized on a substrate by way of a linker molecule. Homobifuctional and heterobifunctional compounds have been devised to link a group present on the reagent to a group present on the substrate. As examples, disuccimidyl suberate and glutaraldehyde are homobifunctional compounds that can covalently bridge an amine group on a reagent molecule to an amine group present on a substrate, such as aminopolystyrene. Additionally, some plastics, such as methyl methacrylate and polyethylvinylacetate, have been developed to bear hydroxyls that can be convened to reactive intermediates. Reactive groups that can be provided include epoxides, hydroxysuccinimide esters, aldehydes, nitrophenyl chloroformate, activated thiols, trityl, tresyl chloride, or other means for reacting free amines, hydroxides or sulfhydryls.
Reagents can be specific nucleic acid sequences. Both DNA and RNA surface coatings can be used to develop novel diagnostic technologies.
Diagnostic tests using nucleic acid sequences have become popular over the past decade since genetic sequence information usually occurs with greater species uniqueness than does protein antigens or isozymes. Diagnostic nucleic acids may be DNA or RNA. Usually the tests employ sample "target" nucleic acids bound to a membrane through ionic and hydrophobic interaction and "probed" by hybridizing a known nucleic acid sequence that has been labeled in some manner. Hybridization of nucleic acid polymers is sequence specific, only two very closely related single nucleic acid chains will form interstrand double strand polymers. When hybridizing nucleic acid polymers the target and probe strands must first be heated to a melting temperature prior to sequence specific annealing. The melting/reannealing temperatures are dependent on the base sequence and length of the strand. In general, short adenine/thymine rich sequences melt more readily than longer strands incorporating guanine/cytosine rich regions. Further, RNA generally melts at temperatures lower than a DNA strand of similar length and base composition.
It is an essential feature of nucleic acid absorption to substrates that the strands must be denatured to efficiently immobilize. Accordingly, mobilization often interferes with the ability of a nucleic acid sequence to hybridize in an assay. DNA is commonly immobilized by absorption to nitrocellulose, nylon, or hyrdoxyapatite. In order for the DNA to remain insolubilized throughout the assay the strand must be of sufficient length to form several attachment points, this form of absorption often slows the ability of immobilized target DNA to efficiently reanneal with labeled probe DNA. Low molecular weight oligonucleotides and RNA are usually immobilized through a process of absorption and covalent attachment to nylon membranes. For covalent attachment to occur amines on the target sequence must be crosslinked to amines on the support. For single strand DNA, RNA and oligonucleotides the free amines of adenine, guanine, and cytosine are often utilized, rendering these bases unavailable for use in forming interstrand diagnostic interactions with labeled probe. Several previous attempts have utilized artificial nucleotides that have free amine groups available for crosslinking. These molecules must be added to the probe strand prior to linking to the amine on the support. One example employs synthetic nucleotide triphosphates with aminoalkyl function groups extending from the base, added to the 5' end of a DNA chain using polynucleotide kinase. This form of an attachment has some use for double strand DNA where amine bearing residues are blocked by interstrand hydrogen bonds, but offers little advantage for single strand DNA and RNA. Covalent crosslinking of polynucleotides to nylon membranes is also accomplished by generation of photoadducts. This process involves the separate steps of absorbing DNA or RNA to a nylon membrane, drying the membrane and using ultraviolet light (usually 254 nm wavelength) to nonspecifically attach bases to the free amines of the nylon. Covalent attachment of nucleotides often renders the DNA useless for diagnostic purposes. For example, crosslinking of amines and carboxylic acids using carbodiimide methods that work well for proteins will render nucleic acids insoluble through interstrand covalent bonds. Aldehydes, such as glutaraldehdye, can attach to hydroxyl groups throughout the length of the polymer's sugar backbone rather than at specific loci, and may crosslink strands together preventing annealing with a labeled probe sequence.
The nonspecific nature of attachment presents difficulties in performing the assay. Probe DNA can bind nonspecifically to the support. To reduce nonspecific "background" binding the hybridization solution must contain a variety of additives, including random lengths of DNA from sources foreign to the target sequences, as well as albumin and polyalcohols. These complex solutions are often sources for error in the diagnostic laboratory, placing severe constraints on the interpretation of data.
The ideal method of nucleic acid immobilization would involve loci specific crosslinking to a support. The loci would be in a portion of the strand that does not participate in hybridization reactions. Further, the ideal support would be a plastic material that is nonporous and has a low surface area to minimize nonspecific interactions with a probe.
Modifications of the plastic surfaces to bear amines, hydroxyls, and sulfhydryls that can be crosslinked or otherwise modified, often results in undesirable characteristics, particularly opacity or decreased structural integrity.
One system that has become available involves incorporation of a methyl imine function. This product requires the end user to convert the methyl imine functionality to a reactive group by addition of crosslinkers (NUNC, Naperville, Ill.). Another system treats plastic with a copolymer of phenylalanine and lysine amino acids to provide a support for a crosslinker (U.S. Pat. No. 4,657,873; Gadow, et al.). Gadow et al. is typical of the other prior attempts at forming reactive surfaces in which reagent immobilization requires several steps and usually entails crosslinking a nucleophile on the reagent molecule with a nucleophile on the plate.
Bienarz et al. (U.S. Pat. No. 5,002,883) also uses an amine bearing surface in combination with a "bridging" molecule to crosslink a reagent molecule to a plastic surface. As does Tetsuo et al. (UK Patent number GB2184127A) which specifically requires hydrophilic functional groups on the surface prior to forming a bond between the reagent and the surface. Packard et al. (U.S. Pat. No. 4,889,916) has a similar requirement for two functional groups to be crosslinked, however, in the case of Packard the reaction is between sulfhydryl groups on both the substrate and the reagent molecule.
The technology of Means et al. (U.S. Pat. No. 4,808,530) produces reagent bearing surfaces by converting hydrophilic groups on proteins to hydrophobic moieties. When the derivatized proteins are contacted to unmodified plastics, the protein is bound by nonspecific adsorption to the surface.
All of the above technologies require a plurality of steps to modify the surface and then crosslink the reagent molecule of interest, or as in the case of Means et al., to modify the protein itself for attachment. Bieniarz et al. describe their derivatization process as requiring several steps over several hours. Typical procedures involve a one hour pretreatment of a prederivatized aminopolystyrene bead, followed by one hour derivatization with several clean up steps; the final step of adding reagent member required overnight incubation. Likewise, Gadow et al. describes a first derivatization step involving heating and mixing, followed by agitation for 30 minutes at room temperature, followed by a 24 hour incubation. At this stage the technology still is incapable of protein binding. The treated plastic resin must be activated for an additional 30 minutes with glutaraldehyde, the actual crosslinking reagent, and washed prior to protein binding.
Means et al. stipulates protein modification prior to binding to a surface. The proteins were modified and purified over the course of several hours, and plastic surfaces were contacted with the protein for an additional several hours. Packard et al. describes labeling of protein species using a heterobifunctional crosslinker. As in Means et al., the Packard technology involves several steps to modify a protein surface, again requiring several hours and extensive purification.
Tetsuo et al. specifies modifying both the protein and the plastic surface. Introduction of thiol groups into proteins required 1 hour plus gel filtration cleanup. Activation of a plastic support required several sequential steps over several hours, plus removal of the reactants. Immobilization of derivatized protein required an additional 24 hours plus cleanup.
The art of polynucleotide identification by sequence specific hybridization assay is well known. U.S. Pat. No. 4,358,535 to Falkow et al. discloses the use of labeled probes in diagnostic reactions. Previous attempts at overcoming the barriers to nucleic acid target sequence immobilization have utilized noncovalent means. Typical of these attempts are Urdea, U.S. Pat. No. 5,200,314. This invention relies on a plurality of steps to isolate a specific target sequence. The technique hybridizes a biotinylated probe to a complementary target sequence, then purifies the target sequence using avidin attached to a column to bind the biotin of the complex. The method leads to a sequence that could be amplified in vitro after desorbing the target nucleotide from the avidin/biotin/capture sequence. Separate technology and apparatus were required for analysis. Some past attempts have utilized specific antibody to DNA, RNA or combinations thereof. Typical of these systems are U.S. Pat. No. 5,200,313 to Carrico using anti-hybrid antibodies, and U.S. Pat. No. 5,273,882 to Snitman employing a complexing agent attached to a nucleic acid probe. Some workers have immobilized oligonucleotides to insoluble substrates using modified copolymers (Sutton et al., U.S. Pat. No. 5,330,891) or proteins and carbohydrates (Warren et al., U.S. Pat. No. 5,328,825). Oligonucleotides attached to fluorescent particles are also described in Brinkley et al., U.S. Pat. No. 5,326,692. Oligonucleotide binding members having delimitative three dimensional configurations have also been employed, as in Lizardi et al., U.S. Pat. No. 5,312,728.
Clearly, there is a need for a simple, rapid system for producing activated surfaces capable of binding nucleic acid reagents in a loci specific manner. In this application we describe a chemical agent capable of producing an activated surface in only a single step. The activated surface is then capable of binding an unmodified nucleic acids without any additional process steps. Further, the availability of nucleic acid surfaces results in novel methodologies for using nucleic acids in diagnosis.